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Full text of "Influence of tumor environment and host immunity on tumor progression and metastasis"

INFLUENCE OF TUMOR ENVIRONMENT AND HOST IMMUNITY 
ON TUMOR PROGRESSION AND METASTASIS 



By 
NANETTE P. PARRATTO 



A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL 

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT 

OF THE REQUIREMENTS FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 

UNIVERSITY OF FLORIDA 

1988 



DEDICATION 
This work is dedicated to my father, for his patience and 
loving support over the many years of its duration; to the 
memory of my mother who taught me well the power of 
perseverance; and to the memory my trusting friend, O'Dee, for 
her unconditional love. 



ACKNOWLEDGEMENTS 
Dr. Arthur Kimura's gentle and persuasive guidance is 
most gratefully acknowledged. He has taught me to think like 
a scientist (we hope) . The constructive criticisms of my 
graduate committee advisors, Dr. Richard T. Smith, Dr. Sigurd 
Nermann, Dr. K. Kendall Pierson, Dr. Maron Calderwood-Mays, 
and Dr. Chris West, are also greatly appreciated. The 
invaluable advice of Dr. Byron Croker, Cynthia Bevis, and 
Norma Houghwout regarding immunohistochemical techniques is 
much appreciated. The contributions of Roberto Luchetta, 
Linda Lee-Ambrose, Janice Odelbralski, Xiang-Hong Tian, and 
Donald Dugger toward the completion of this work is also much 
appreciated. 



111 



TABLE OF CONTENTS 

page 

ACKNOWLEDGEMENTS iii 

LIST OF TABLES vi 

LIST OF FIGURES vii 

KEY TO ABBREVIATIONS ix 

ABSTRACT X 

INTRODUCTION AND REVIEW OF LITERATURE 1 

MATERIALS AND METHODS 4 

Mice 4 

Murine Melanoma Cell Lines 4 

Other Murine Cells and Cell Lines 6 

Monoclonal Antibodies 7 

Affinity Purification of Monoclonal Antibody for 

Biotin and Fluorescein Isothiocyanate 

Conjugation 7 

Flow Cytometry 9 

Subcutaneous Tumor Generation and Spontaneous and 

Experimental Metastasis Assays 9 

Immunocytochemistry, Immunohistology and Direct 

Immunofluorescence 10 

Immunization Protocols 12 

Radioimmunologic Determination of MoAb and Immune 

Sera Binding Indices 13 

Immuno-magnetic Bead Separation of Immunoreactive 

Subpopulations 14 

Cell Solubulization, Polyacrylamide Gel 

Electrophoresis and Western Blotting 14 

Statistical Analyses 15 

RESULTS 16 

Anti-Met-72 Monoclonal Antibody Binding to Fixed 

Melanoma Cells 16 

Optimum Biotin Substitution of Monoclonal Antibody 16 

Biotin Conjugated Anti-Met-72 Monoclonal Antibody 

Specificity 17 



IV 



Fluorescence Activated Cell Sorting Selection of 

Met-72 Positive Variants Isolated From a Fresh 

Experimental Ovarian Metastasis 17 

Bone Marrow Derived Clones of B16 Melanoma Express 

High Levels of Met-72 18 

Immunocytology of Fresh Ovarian and Lung Metastases. .. .18 
Localized Distribution of Met-72 Positive Variants 

Within Progressing Subcutaneous Melanoma 19 

Met-72 Positive Variants Localized Within B16 

Melanoma Metastases 20 

Specificity Characteristics of Syngeneic Antibodies 

Generated Against B16 Melanoma 20 

Syngeneic Antibody Response Against a C3H Melanoma 23 

Metastatic Potential of Melanoma Populations 

Negatively Selected By Syngeneic 

Anti-Melanoma Antibodies 23 

Biochemical Characterization of Anti-B16-Fl 

Reactive Species 25 

In Situ Characterization of Metastatic Variants Within 

Experimental Metastases and Subcutaneous Masses of 

B16 Melanoma 25 

Immunohistology of Syngeneic Healing Skin Wounds and 

Embryos 28 

DISCUSSION 79 

REFERENCES 91 

BIOGRAPHICAL SKETCH 100 



LIST OF TABLES 

page 

Table 1. Results of Lung Colonization Assays 29 

Table 2. Syngeneic Immune Sera Reactivity with C57BL/6 

Melanomas 49 

Table 3. Syngeneic Immune Sera Reactivity with C57BL/6 

Melanomas 51 

Table 4. Results of Analysis of Variance Comparing Groups 
of Mice Injected with Negatively Selected B16 
Melanoma Cells 64 

Table 5. Level of Monoclonal Antibody Binding to Cryostat 

Sections of Subcutaneous B16 Melanomas 72 



VI 



Figure 1 . 

Figure 2 . 
Figure 3 . 
Figure 4 . 

Figure 5. 

Figure 6. 
Figure 7. 

Figure 8 . 
Figure 9. 

Figure 10. 

Figure 11. 
Figure 12. 



LIST OF FIGURES 

Page 

Radiolabeled anti-Met-72 monoclonal antibody 
binding to fresh or ethanol fixed C57BL/6 
melanoma cells 31 

Optimum biotin conjugation of anti-Met-72 
monoclonal antibody 33 

Retention of binding specificity of anti-Met-72 
MoAb after biotin conjugation 35 

Flow cytometric analysis of Met-72 positive 
metastatic variants within a fresh ovarian 
metastasis of B16-F1 37 

Clones derived from experimental metastases to 
ovaries retain a high expression of Met-72 upon 
repeated cycling in vivo 39 

Anti-Met-72 monoclonal antibody binding to clones 
of bone marrow derived melanoma metastases 41 

Met-72 positive variants of B16 melanoma detected 
in cytospin and impression smear preparations of 
metastases 43 

Localization of Met-72 positive variants within 
developing B16 subcutaneous melanoma 45 

Direct immunofluorescence of Met-72 positive 

variants localized within B16 melanoma 

metastases 47 

Flow cytometric analysis of anti-B16-Fl immune 
sera (A) and anti-B16-Fl monoclonal antibody (B) 
binding to C57BL/6 melanomas 53 

Anti-B16-Fl monoclonal antibody binding to a panel 
of murine cells 55 

Syngeneic antibody response against K1735 
melanoma 57 



VII 



Figure 13. 

Figure 14. 

Figure 15. 
Figure 16. 

Figure 17. 

Figure 18. 

Figure 19. 
Figure 20. 

Figure 21. 

Figure 22. 



Flow cytometric analysis of B16-F1 cells before 
and after immunomagnetic bead selection and 
removal of reactive subpopulations 59 

Flow cytometric analysis of B16 melanoma before 
and after immunomagnetic bead selection and 
removal of reactive subpopulations 61 

Experimental metastatic activity of negatively 
selected B16 melanoma cells 63 

Western blot analysis of anti-B16-Fl immune sera 
and MoAb binding to lysates of syngeneic melanoma 
metastatic variant cell lines 65 

Immunocytology of metastatic variants exfoliated 
from fresh experimental lymph node metastases of 
BL6-10 67 

Localization of B16 melanoma metastatic variants 
within metastases by immunoperoxidase 
cytochemistry 69 

Localization of B16 melanoma variants within 
experimentally colonized lungs 71 

Localization of B16 melanoma variants within 
subcutaneous tumors maintained in normal syngeneic 
hosts 74 

Localization of B16 melanoma variants within 
subcutaneous tumors maintained in 500 Gy 
irradiated syngeneic hosts 76 

Localization of B16 melanoma variants within 
subcutaneous tumors maintained in allogeneic 
athymic hosts 78 



vm 



KEY TO ABBREVIATIONS 
aminoethylcarbazole (AEC) 
antibody (Ab) 

avidin-biotin-horseradish peroxidase complex (ABC) 
counts per minute (cpm) 
Eagles Hank's amino acid media (EHAA) 
fetal bovine serum (FBS) 
fluorescein isothiocyanate (FITC) 
fluorescein isothiocyanate conjugated F(ab')2 sheep anti-mouse 

IgG (FITC-SAM) 
flow cytometry (FACS) 
Gray (Gy) 

intraperitoneally (i.p.) 
intravenous (i.v.) 
monoclonal antibody (MoAb) 
newborn calf serum (NCS) 
normal mouse serum (NMS) 
phosphate buffered saline (PBS) 
phosphate buffered saline with 0.7 mM EDTA and 0.6 mM glucose 

(cPEG) 
protein A (pA) 
radioimmunoassay (RIA) 
streptavidin (sA) 
subcutaneous (s.c.) 

ix 



Abstract of Dissertation Presented to the Graduate School 

of the University of Florida in Partial Fulfillment of the 

Requirements for the Degree of Doctor of Philosophy 

INFLUENCE OF TUMOR ENVIRONMENT AND HOST IMMUNITY 
ON TUMOR PROGRESSION AND METASTASIS 

By 

Nanette P. Parratto 

December 1988 

Chairman: Arthur K. Kimura 
Major Department: Pathology 

Monoclonal antibodies (MoAb) against Met-72, a 72,000- 

dalton cell surface glycoprotein, have been used to 

characterize B16 melanoma variants expressing high 

experimental metastatic activity. Freshly isolated bone 

marrow and ovarian metastases of B16 melanoma were found to 

express high levels of Met-72 by radioimmunoassay (RIA) . Lung 

and ovarian metastases were shown immunocytochemically to 

contain melanoma cells with heterogeneous levels of Met-72 

antigen. Flow cytometric analysis (FACS) of an ovarian 

metastasis revealed a discrete subpopulation of positive 

cells. Clones derived from this ovarian metastasis were found 

to express high levels of Met-72 and high experimental 

metastatic potential to ovaries. Finally, Met-72 positive 

variants were localized by routine immunohistology within 

developing metastatic and subcutaneous B16 melanomas. The few 



cells which showed high intensity Met-72 staining have been 
consistently observed to be within microscopic foci in 
colonized organs and positioned within regions of 
subcutaneous masses. 

Melanoma has been shown to be immunogenic in experimental 
and clinical situations; however, syngeneic antibodies (Ab) 
against highly metastatic variants have only been obtained by 
experimental manipulation of the host immune response. 
Binding of anti-B16 melanoma Ab elicited by syngeneic 
intravenous immunization was inversely correlated with 
metastatic activity of melanoma lines and clones by RIA. Flow 
cytometric analyses similarly revealed that the proportion of 
reactive cells in melanoma populations was inversely 
correlated to their experimental metastatic activity. Anti- 
B16 melanoma MoAb derived by hybridoma technology from 
intravenously challenged mice, showed distinct anti-melanoma 
specificity in RIA. Monoclonal antibody directed against non- 
metastatic melanoma variants defined specific bands in Western 
blot analyses of Mr 45,000 and 50,000. Finally, negative 
cellular selection technigues revealed an increased 
experimental metastatic potential associated with the non- 
immunogenic B16 melanoma subpopulation. 

The results of these experiments suggest that 1) Met-72 
antigen expression may be a common surface phenotype of B16 
melanoma metastatic variants; 2) microenvironmental factors 
may be instrumental in the induction, attraction or 

xi 



maintenance of metastatic variants, and 3) the host antibody 
response may be a key factor in the regulation of levels of 
metastatic variants during tumor progression by selectively 
targeting poorly metastatic populations. 



xn 



INTRODUCTION AND REVIEW OF LITERATURE 
The dynamics of tumor cell heterogeneity is clearly 
evident from clinical and experimental studies of tumor 
progression (1,2). The diagnosis and treatment of solid 
tumors has been seriously impaired by a poor understanding of 
the clonal evolution of heterogeneous populations within 
developing tumors (3,4). Experimental evidence documents the 
existence of subpopulations of metastatic tumor cell variants, 
exhibiting a range of metastatic potentials from low to high 
(3-6) . The precise nature and expression of the phenotypic 
characteristics during the natural history of metastasis 
remains unique to each tumor bearing host and the particular 
solid tumor (7) . Clearly, classical pathologic descriptions 
have been inadequate to identify unique metastatic variants 
within surgical specimens. This remains a hindrance to 
quantitative classification of metastatic potential and 
patient prognosis. 

Paulus et al. (8) have implicated regional variations of 
differentiation in B16 melanoma, localized by morphologic 
heterogeneity. Direct, in situ visualization of metastatic 
tumor cell variants has not been possible until recently. We 
have identified a Mr 72,000 cell surface glycoprotein (Met-72) 
quantitatively associated with highly metastatic tumor cell 



2 
variants of the B16 melanoma (5). More recently, a Mr 83,000 
native form of this surface molecule (Met-72/83) has been 
described (9) . The experimental metastatic potential of over 
30 B16 melanoma clones has been correlated to a quantitative 
surface expression of Met-72 (5,10,11). In addition, 
fluorescence activated cell sorting (FACS) has now been used 
to directly isolate metastatic variants from the heterogeneous 
parental B16-F1 tumor (12) . 

The present study was designed to visualize, isolate and 
histologically localize metastatic variants present in fresh 
B16 melanoma tumor masses. Anti-Met-72 monoclonal antibodies 
(MoAb) reveal a unique localization of Met-72 positive cells 
along the invading front of the progressing tumor. These 
experiments show that anti-Met-72 MoAb previously used to 
characterize the expression of Met-72 in vitro , may also be 
useful for isolation and localization of highly metastatic 
variants in situ in progressing primary and metastatic B16 
melanoma. 

Much of the success in defining antigenic systems for 
melanoma is related to the immunogenicity of this tumor type. 
Syngeneic and autochthonous antibody (Ab) responses against 
melanoma have been demonstrated in a number of experimental 
(13-20) and clinical situations (21-24) , and yet in syngeneic 
combinations, Ab against highly metastatic variants have only 
been obtained through experimental manipulation of the host 
immune response (5) . Taken together, these results suggest 



3 
that the immunodominant melanoma population may in fact be 
those cells with a low metastatic activity. 

The current view that tumors are comprised of 
heterogeneous subpopulations with widely varying and changing 
metastatic potentials, has been supported in a number of tumor 
systems (3,4). Selective host responses against the more 
poorly metastatic subpopulations could thus be expected to 
dramatically influence the course of tumor growth, 
progression, and metastasis. 

Experiments described here are focused at characterizing 
the range of Ab specificities elicited by metastatically 
distinct cell lines and clones of murine melanoma and the 
potential impact of such responses on tumor progression and 
metastasis. Antisera and MoAbs derived in these studies have 
been used to fractionate melanoma populations and deduce the 
metastatic potentials of the target population. Negatively 
selected melanoma cells are shown to have enhanced metastatic 
activity after removal of the anti-melanoma reactive 
population. These findings strongly support the conclusion 
that subpopulations of poorly metastatic melanoma are primary 
targets of host humoral immunity. The relationship of these 
findings to other anti-melanoma Ab is discussed. 



MATERIALS AND METHODS 
Mice 

C57BL/6, C3H/HeJ, and Balb/c nu/nu mice were purchased 
from the Jackson Laboratory (Bar Harbor, ME) and maintained in 
the Tumor Biology Unit mouse colony, Department of Pathology, 
in accordance with the National Institutes of Health 
Guidelines for the Use of Experimental Animals. Female mice, 
8 to 16 weeks of age were used in these studies. C57BL/6 mice 
were exposed to 500 Gy total body irradiation. 

Murine Melanoma Cell Lines 

Early passages of the B16-F1, B16-F10 and B16-BL6 
derivatives of the C57BL/6 melanoma, B16, were obtained from 
the Division of Cancer Treatment Tumor Bank (E. G. and G. 
Mason Research Institute, Worchester, MA) where they had been 
deposited by Dr. I. J. Fidler. A recently derived syngeneic 
melanoma, JB/RH, was provided by Dr. Jane Berklehammer (AMC 
Cancer Research Center, Denver, CO) (25,26) for comparison in 
our studies. Clones of the B16 melanoma were derived by 
limiting dilution and micromanipulation (5) from both the 
poorly metastatic B16-F1 cell line and the in vitro selected, 
highly invasive metastatic variant, B16-BL6 (27) . Stocks from 
early passages of all cell lines and clones were frozen at 
-70 °C and restarted every 8 to 12 weeks to limit the 



5 
possibilities of functional and phenotypic drift. All cell 
lines and clones were maintained in vitro at 37 °C in a 
humidified incubator containing 8% C0 2 , by subculturing every 
4 days. Monolayers of cells were detached from the petri 
dishes (Costar #3100, Cambridge, MA) by a 3-minute room 
temperature incubation with 136 mM NaCl, 3 mM KC1, 1.5 mM 
KH 2 P0 4 , 8 mM Na 2 HP0 4 , 0.7 mM EDTA and 0.6 mM glucose (cPEG) 
(28) . For routine passage, cells were washed and replated at 
a concentration of 5 X 10 5 cells/10 cm dish in 10 ml media. 
The poorly metastatic B16-F1 parental line and highly 
metastatic B16-F10 line were maintained in Cellgro MEM 
(Sybron, Washington, D.C.) supplemented to 10% with heat 
inactivated fetal bovine serum (Grand Island Biologicals 
[GIBCO], Grand Island, NY), 100 U/ml penicillin, 100 jig/ml 
streptomycin, 1 mM pyruvate, 2 mM glutamine and 27 mM NaHC0 3 . 
The highly metastatic clone BL6-10, derived from B16-BL6, 
clones derived by limiting dilution from extrapulmonary 
metastases of B16-F1, and the poorly metastatic line, JB/RH, 
were maintained in Hanks' balanced salt solution, minimal 
essential media amino acids, nonessential amino acids and 
vitamin solution (GIBCO), and nucleosides (Sigma, St. Louis, 
MO) (EHAA) supplemented to 10% with newborn calf serum 
(GIBCO) , 100 U/ml penicillin, 100 Mg/ml streptomycin, 18 mM 
NaHC0 3 and 0.05 mM 0-mercaptoethanol (29). 



6 
Other Murine Cells and Cell Lines 
A panel of murine cells and cell lines was assembled for 
specificity analysis of the anti-B16-Fl antisera and MoAb 
secreting hybridomas captured by the immunization procedures. 
Syngeneic tumor cell lines included PAK17 and PC8 
(methylcholanthrene induced fibrosarcomas) , Lewis lung 
carcinoma, EL-4 T-cell lymphoma, and normal syngeneic lung 
fibroblasts (provided by Dr. Paul Klein, Department of 
Pathology, University of Florida) . An allogeneic B cell 
lymphoma, 2PK3 (Balb/c origin) was also obtained from the 
Department of Pathology, Tumor Bank. These cell lines were 
maintained in Dulbecco's modified minimum essential media 
(GIBCO) supplemented to 10% with FBS (GIBCO) , 100 U/ml 
penicillin, 100 fxq/ml streptomycin, 1 mM pyruvate and 2 mM 
glutamine (5) . An allogeneic UV induced melanoma, K1735 
(C3H/HeN origin) (30) was obtained from the Department of 
Pathology, Tumor Bank and maintained in 10% newborn calf serum 
supplemented EHAA. The P815 mastocytoma of the DBA/2 strain 
(provided by Dr. Sigurd Normann, Department of Pathology, 
University of Florida) , was maintained in RPMI (Gibco) 
supplemented to 10% with FBS plus 100 fig/ml streptomycin, 100 
U/ml penicillin. Cells were harvested during log phase of 
growth for use in radioimmunoassays (RIA) . Single cell 
suspensions of normal C57BL/6 and Balb/c spleen cells were 
prepared by mechanical disaggregation and lysis of RBC in 17 
mM Tris, 144 mM NH 4 C1, pH 7.2. 



7 

Monoclonal Antibodies 

The anti-B16-Fl monoclonal antibodies (MoAb) derived in 

these studies were generated as described (5,31). Briefly, 

spleen cells from mice injected i.v. with 3 X 10 5 B16-F1 cells 

were fused with BALB/c myeloma SP2/0-Agl4 cells and dispensed 

under limiting dilution conditions into 96 well plates. 

Supernatants from growth positive wells were screened by RIA 

for binding to the poorly metastatic melanoma line, JB/RH. 

The highest binding hybridomas were subcloned and screened for 

specificity using JB/RH and BL6-10 cells. Monoclonal antibody 

isotypes were determined by enzyme linked immunosorbant assay 

(Southern Biotechnology Associates, Inc., Birmingham, AL) . 

Monoclonal antibodies derived by syngeneic immunization of 

C57BL/6 mice with B16-F1 melanoma (anti-B16-Fl MoAb; 2D8.3B1 

and 2H5.1B1, designating clone and subclone) were both found 

to IgG 3 : kappa. Another syngeneic anti-B16-Fl MoAb (1F11.1E12) 

was found to be IgG 2a : kappa. Anti-Met 72 MoAb were generated 

by syngeneic immunization of C57BL/6 mice with selected B16 

melanoma clones. The specificity and characteristics of these 

MoAbs have been reported in detail (5,31). Hybridoma cells 

secreting an isotype identical, negative control MoAb used in 

this study (anti-sheep red blood cell, N-S.7, IgG3: kappa) were 

obtained from the American Type Culture Collection. 

Affinity Purification of Monoclonal Antibody for 
Biotin and Fluorescein Isothiocvanate Conjugation 

Monoclonal antibodies were affinity purified by 

fractionation through protein A-Sepharose 4B (Pharmacia, 



8 

Piscataway, NJ) (32) and purity checked by sodium dodecyl 
sulfate 10% polyacrylamide gel electrophoresis (33) . Affinity 
purified IgG (1 mg/ml) was dialyzed against 100 mM NaHC0 3 , 200 
mM NaCl pH 8.2 and then reacted with 2 mM sulfosuccinimidyl 6- 
(biotinamido)hexanoate (Pierce Chemical, Rockford, IL) in 
dimethyl formamide (Sigma) , at biotin ester: protein ratios of 
1:2.5 to 1:10 (weight/ weight) for 4 hours at room temperature, 
in the dark. The reaction was stopped by the addition of 1 M 
NH 4 C1 to a final concentration of 100 mM in the reaction 
mixture. Unreacted biotin was then removed by exhaustive 
dialysis against PBS. Biot in-conjugated protein 
concentrations were determined by optical density at 280 nm 
(34) . Optimal biotin ester/protein ratios used for the 
conjugation of the various preparations were determined 
empirically by flow cytometric analysis on cell preparations 
(35) . Briefly, 2 X 10 6 melanoma cells were incubated with 
biotin conjugated antibody at 4°C for 30 min. in the dark. 
After 3 washes in PBS supplemented to 2% with FBS, either 
native or biotin conjugated MoAb were incubated with 
fluorescein isothiocyanate conjugated F(ab')2 sheep anti-mouse 
IgG (Cooper Biomedical, Malvern, PA) (FITC-SAM) or FITC 
conjugated avidin (Vector Laboratories, Burlingame,CA) . After 
3 washes with PBS, 1 X 10 4 cells were counted and analyzed by 
flow cytometry (12,35). 

Fluorescein isothiocyanate (FITC) conjugation was as 
described by Goding (36) . Briefly, affinity purified MoAb 



9 

were dialyzed against 80 mM Na 2 C0 3 , 200 mM NaHC0 3 pH 9.5 and 

reacted with 2 mg FITC per ml dimethylsulfoxide (Sigma) at a 

FITC/protein molar ratio of 4 . Unreacted FITC was separated 

by desalting through Sephadex G-25 (Pharmacia) . 

Flow Cytometry 

Cultured cell lines were harvested as described for 

routine passage and washed twice in ice cold PBS. Aliguots of 

10 6 cells were incubated at 4°C for 45 min. with diluted 

normal or immune serum or with free or conjugated MoAb as 

described in the results section. After two washes with 100 

volume excess of ice cold PBS, labeled cells were 

counterstained with FITC-SAM for 45 minutes at 4°C. Labeled 

and stained cells were washed, resuspended at 5 X 10 5 cells 

per ml and filtered through 41jx nylon mesh (Spectrum, Los 

Angeles, CA) . Flow cytometric analysis was performed with the 

FACSTAR argon laser (Becton/Dickinson, Oxnard, CA) the 

photomultiplier voltage for green fluorescence set at 400, the 

amplifier gain set at log scale and the photodiode for forward 

scatter set at for sample analyses. Data was collected and 

analyzed by the Becton/Dickinson Consort 30 computer program 

(Oxnard, CA) (35) . 

Subcutaneous Tumor Generation and 
Spontaneous and Experimental Metastasis Assays 

Single cell suspensions of B16-F1, B16-F10 or BL6-10 

metastatic variant lines were injected s.c. (2 X 10 6 cells per 

0.1 ml 136 mM NaCl, 3 mM KC1, 1.5 mM KH 2 P0 4 , 8 mM Na 2 HP0 4 

[PBS]) into the caudal mid-dorsum of age matched normal or 500 



10 
Gy irradiated C57BL/6 mice or into Balb/c nu/nu mice, to 
generate primary tumor masses. Animals were anesthetized with 
50 mg/kg Na pentabarbital i.p. to aseptically excise tumors 
and associated skin at various intervals of tumor growth; the 
wounds were then clipped. Spontaneous metastasis formation 
was assessed by sacrifice and necropsy 21 days post- 
operatively. Experimental metastasis of all melanoma lines 
was assessed by sacrifice and necropsy 21 days after lateral 
tail injection of 3 X 10 5 cells/0.2 ml PBS into age matched 
syngeneic female mice. 

Immunocytochemistry. Immunohistology and 
Direct Immunofluorescence 

Cytospin and impression smear preparations of freshly 

excised B16 melanoma tumor masses were examined for Met-72 

expression by immunoperoxidase staining. Single cell 

suspensions of tumor masses were generated by teasing tissue 

to 1 mm 3 pieces followed by an 8 to 10 hour incubation at 4°C 

in 10 ml/gm tumor weight, EHAA supplemented to 0.1% with 

collagenase V (Sigma) (37) . Viable cells were separated on 

Ficoll Hypaque (Pharmacia) , washed, and endogenous peroxidase 

activity blocked by 30 min. incubation in PBS supplemented to 

5% with heat inactivated FBS and 0.3% with H 2 2 at 4°C. Cells 

were labeled in the dark with biotin conjugated MoAb for 30 

min. at 4°C, washed and incubated with avidin-biotin- 

horseradish peroxidase complex ([ABC] Vector Laboratories, 

Burlingame, CA) for 30 min. at 4°C. Approximately 3 X 10 4 

labeled cells were cytocentrifuged onto poly-D-lysine coated 



11 

microslides, air dried, fixed in acetone for 30 seconds and 
rehydrated in PBS. The reaction was developed with 
aminoethylcarbazole ([AEC] Sigma) in the presence of H 2 2 . 
The slides were washed and counter stained with Harris' 
hematoxylin (Sigma) (38,39). 

Freshly excised tumor masses, rinsed in ice cold PBS, 
were gently touched to coated slides to create impression 
smears. Slides were air dried, fixed in acetone for 10 min. , 
rehydrated with PBS and either immediately processed by 
immunohistology or air dried and stored at -70 "C for future 
use. Impression smears provide a rapid screening tool for 
detection of antigen expression in vivo (40,41). 

B16 melanoma tumors from subcutaneous or experimental 
metastatic lesions were excised, snap frozen in isopentane 
chilled with liguid nitrogen and stored at -70 °C until 
sectioned. Subcutaneous masses were examined after 5 to 21 
days of growth; metastatic lesions after 14 to 21 days of 
growth. Skin wounds created surgically on the mid-dorsum were 
excised at various points during healing, snap frozen and 
stored at -70°C. C57BL/6 embryos were collected at days 11 
and 15 of gestation, snap frozen and stored at -70 °C. 

Cryostat sections 2 to 12 urn thick were stained according 
to a modification of described technigues (42,43). Briefly, 
air dried, acetone fixed, sections were flushed with 0.01% 
avidin (Vector Laboratories) for 15 min. and then 0.134 mM 
biotin (Sigma) for 15 min. with a PBS wash between. Sections 



12 
were then blocked for endogenous peroxidase activity as for 
cytospin preparations. Biotin conjugated MoAb at 
predetermined optimal concentrations were layered onto 
sections and allowed to react for 30 min. in a dark, 
humidified chamber. Slides were washed with 20 volume excess 
PBS and reacted with the ABC reagent for 30 min. The reaction 
was developed with AEC, the slides counterstained with 
hematoxylin and examined under oil immersion. 

Frozen sections were similarly air dried, acetone fixed 
and rehydrated with PBS for direct immunofluorescence using 
FITC conjugated MoAb. Slides were washed with PBS, 
coverslipped and examined under high power using a 490 nm 
excitation filter and 510 nm emission filter for green 
fluorescence . 

Immunization Protocols 

Age matched female mice were injected via the tail vein 
with in vitro maintained melanoma lines after the cells had 
been lifted with cPEG and washed three times with ice cold 
PBS. Mice (4 to 6 per experimental group) were immunized with 
various numbers of cells and immune sera was harvested at 
various times to determine dose response and kinetics curves. 
Optimal dosages and response times were determined and used to 
further characterize the serologic immune response. Normal 
mouse serum (NMS) harvested from age matched littermates 
served as background control. Following exsanguination, 
animals were killed by cervical dislocation and then 



13 

necropsied. Systemic experimental metastatic colonization was 

assessed by counting lung and extrapulmonary tumor nodules 

after fixation of the organs in buffered formalin (6) . 

Radioimmunologic Determination of MoAb and 
Immune Sera Binding Indices 

MoAb binding to the various cell types was measured 1) 

directly using radiolabeled MoAb (44), or 2) indirectly with 

125 I labeled protein A as described (45) or with 125 i labeled 

streptavidin (Zymed Laboratories, Inc. , San Francisco, CA) for 

biotin conjugated MoAb using a modification of Philpott, et 

al. (46) . Briefly, 2 X 10 5 freshly harvested cultured cells 

were incubated for 45 minutes at 4°C with equivalent dilutions 

of normal or immune mouse serum or MoAb. Washed cells were 

incubated for an additional 45 minutes with 2 X 10 5 cpm 

radiolabeled protein A or streptavidin. Radioactivity 

remaining bound to the cells after washing was assessed by 

gamma scintillation counting in an LKB gamma counter (LKB 

Instruments Inc., Gaithersburg, MD) . Specific MoAb or immune 

sera binding to the different cell types is expressed as mean 

± S.D. cpm bound or as a binding index (47), calculated from 

duplicate determinations as follows: 

mean cpm bound with specific 

MoAb or immune sera 

Binding index = 

mean cpm bound with 

control MoAb or NMS 



14 

This normalizes individual cell line differences in background 

binding and allows comparison between the different cell 

types . 

Immunomagnetic Bead Separation of Immunoreactive Subpopulations 

Immunomagnetic beads (Dynabeads M-450, Dynal Inc., Fort 

Lee, N.J.) coated with sheep anti-mouse IgG were used to 

separate cells which had been incubated with normal versus 

anti-B16-Fl immune sera or anti-sheep RBC MoAb versus anti- 

B16-F1 MoAb. Cells were harvested from culture, washed and 

resuspended in hybridoma exhaustion supernatant or PBS 

supplemented to 5% with normal or immune serum, and incubated 

for 45 minutes on ice. Immune labeled cells were washed and 

then incubated with immuno-magnetic beads at a bead to target 

cell ratio of 3 to 1 for 10 minutes at 4°C. Cells bound to 

beads were separated from those cells remaining free in 

suspension by applying a cobalt steel magnetic force (Dynal 

MPC-1, Dynal Inc., Fort Lee, N. J.) for 1 minute (48). This 

process was repeated once again with the non-bound cells. The 

remaining negatively fractionated cells were analyzed by flow 

cytometry. A portion was injected intravenously at 3 X 10 5 

cells per 0.2 ml PBS to assess experimental metastatic 

capacity. 

Cell Solubilization. Polvacrvlamide Gel Electrophoresis and 

Western Blotting 

Cell lines maintained in vitro were lifted with cPEG, 

washed in PBS and solubilized in PBS supplemented to 0.5% with 

Nonidet P-40 [ (NP-40) Bethesda Research Laboratories, 



15 
Gaithersburg, MD] , 1 % epsilon amino caproic acid and 1 mM 
phenylmethylsulfonyl fluoride (Sigma Chemical Co., St. Louis, 
MO) at 4"C for 30 min. Supernate was recovered by 
centrifugation of the detergent lysate for 15 min. at 12,000 X 
g, 4°C (49,50). Lysates and marker proteins (Rainbow Markers, 
Amersham, Arlington Heights, IL) were electrophoresed on 5 to 
15% or 10% acrylamide slab gels under reducing conditions 
(33) . Proteins were electro-blotted to polyvinyl idene 
difluoride solid membrane (Immobilon, Millipore Corp., 
Bedford, MA) as described (51,52) and immuno-stained with 
anti-B16-Fl antisera or anti-B16-Fl MoAb. Antibody bound 
protein was visualized by protein A conjugated peroxidase 
catalysis of H 2 2 in the presence of diaminobenz idene (39) . 

Statistical Analyses 
Significance was determined by analysis of variance, 
computed using the SAS program (Biostatistics, J. Hillis 
Miller Health Center, University of Florida) . 



RESULTS 

Anti-Met-72 Monoclonal Antibody Binding to 
Fixed Melanoma Cells 

The highly metastatic B16 melanoma variant, BL6-10, or 

syngeneic poorly metastatic line, JB/RH (Table 1) , were 

reacted against radiolabeled anti-Met-72 MoAb. Radioactivity 

remaining bound to fresh or 95% ethanol fixed cells in 

suspension is shown in Figure 1. The highly metastatic clone, 

BL6-10, binds eguivalently high levels of Ab whether fresh or 

fixed. Fixation of the poorly metastatic line JB/RH does not 

artificially increase binding of anti-Met-72 MoAb. 

Optimum Biotin Substitution of Monoclonal Antibody 

Biotin conjugation to proteins is an empiric reactiion, 

the optimum conditions varying uniquely for each protein (34) . 

Affinity purified anti-Met-72 or control MoAb were reacted 

with the biotin ester at varying weight/weight ratios. Flow 

cytometric analysis of the binding of three different 

preparations to BL6-10 is shown in Figure 2. The positive 

fluorescence under curve C in Figure 2 B is comparable to the 

binding of affinity purified MoAb in Figure 2 A. This ratio 

of 8 to 1 (protein to biotin) was also found to be optimal for 

maintenance of control MoAb negative fluorescence. 



16 



17 

Biotin Conjugated Anti-Met-72 Monoclonal Antibody Specificity 

Reactivity of biotin conjugated MoAb against the highly 

metastatic B16 melanoma clone, BL6-10, is compared in Figure 3 

to the poorly metastatic parental B16-F1 melanoma and to a 

syngeneic, non-metastatic melanoma, JB/RH (Table 1) . There is 

approximately a five-fold lower level of anti-Met-72 MoAb 

binding by RIA to the poorly metastatic cell lines. This 

result documents a retention of specificity in binding to the 

highly metastatic clone after biotin conjugation. 

Fluorescence Activated Cell Sorting Selection of Met-72 

Positive Variants Isolated From a 

Fresh Experimental Ovarian Metastasis 

Experimental metastases from the parent B16-F1 melanoma 

were generated as described. An ovarian metastasis was noted 

after 21 days and removed for analysis of Met-72 expression by 

flow cytometry. Biotin conjugated anti-Met-72 MoAb revealed 

two distinct populations within the freshly excised ovarian 

metastasis, one highly Met-72 positive (Figure 4) . Melanoma 

cells sorted from this population and maintained in vitro have 

retained a high binding profile to anti-Met-72 MoAb (data not 

shown) . Cells obtained from the original ovarian metastasis 

(0-1) were cultured, tested by RIA for Met-72 expression 

(Figure 5, selection cycle 1) , cycled in vivo to generate 

experimental metastases and cloned by limiting dilution to 

obtain 0-1.1 (Figure 5). Clone 0-1.1 from the original 

ovarian metastasis was tested for binding to anti-Met-72 MoAb 

(Figure 5, selection cycle 2) and 3 X 10 5 cells were injected 



18 
i.v. in the syngeneic host, resulting in the organ selectivity 
indicated in Figure 5, selection cycle 2. Subseguent ovarian 
metastases were isolated, cultured, tested for Met-72 
expression and cycled in vivo (Figure 5, 0-1.2, cycle 3 and 0- 
1.3, cycle 4). Successive cycles of in vivo passage and 
culturing were seen to enhance organ selectivity for ovaries 

(53%, 56%, 87% of the mice having ovarian metastases) and to 
enrich for Met-72 expression (28, 36, 45 times background) 

(Figure 5) . 

Bone Marrow Derived Clones of B16 Melanoma 
Express High Levels of Met-72 

Experimentally derived bone marrow metastases of B16-F1 

melanoma were aspirated and mechanically disaggregated into a 

single cell suspension. Aspirates from five animals were 

cloned by limiting dilution into 96 well plates. Growth 

positive wells were expanded in culture and tested for binding 

to anti-Met-72 MoAb. Clones of bone marrow metastases from 

each of the five animals are shown in Figure 6, compared to 

BL6-10 and the parental B16-F1 cells. As seen with the 

ovarian metastases, Met-72 expression of clones derived from 

bone marrow metastases was increased above the parental B16-F1 

level of expression, and in some cases approached that of the 

highly metastatic BL6-10 clone. 

Immunocytology of Fresh Ovarian and Lung Metastases 

B16 melanoma metastatic variants were isolated from fresh 

lung and ovarian experimental metastases by mechanical and 

enzymatic disaggregation. Single cell suspensions were 



19 

processed for immunocytology using anti-Met-72 MoAb. Cytospin 

preparations of an experimental ovarian metastasis showed 

positive immunoperoxidase staining (Figure 7 B) . There is 

cytomorphologic heterogeneity in the level of expression of 

Met-72 within this mixed cell population. Impression smears 

of experimental and spontaneous lung metastases generated by 

injection of the poorly metastatic Fl parental B16 melanoma 

demonstrated similar levels of individual cellular binding to 

anti-Met-72 MoAb (Figure 7 D, F) . Isotype identical MoAb 

(anti-sheep RBC, N-S.7) showed no staining in these assays 

(Figure 7 A, C, E) . 

Localized Distribution of Met-72 Positive Variants 
Within Progressing; Subcutaneous Melanoma 

Immunohistologic analyses of cryostat sections from s.c. 

B16-F1 parental and BL6-10 melanoma were performed at various 

time points during tumor growth. Predetermined optimal 

concentrations of biotin conjugated MoAb were incubated with 

serial sections of snap frozen tumor. Reactive sites were 

detected as red granules upon development with AEC. A common 

pattern of reactivity has been noted in all sections examined 

from tumors as early as 3 days to as late as 15 days of 

growth. A subcutaneous B16-F1 derived tumor of 7 days growth 

serially sectioned and stained with anti-Met-72 MoAb is shown 

in Figure 8. Background levels of peroxidase staining are 

shown in Figure 8 A and B, using an isotype identical 

biotinylated control MoAb, N-S.7. In contrast, Met-72 

positive cells were observed to be localized within regions of 



20 

the tumor mass (Figure 8 C, D, F) . The most brightly Met-72 

positive cells were seen within satellites which appear to 

advance from the main tumor mass. Morphologically, these 

cells are variably melanotic. Early observations showed a red 

reaction product around vascular areas (Figure 8 E) . A 

definitive association of this Met-72 positive staining with 

melanoma cells has not been reproducible. Notably, no binding 

was seen in the necrotic areas of the tumor mass. 

Met-72 Positive Variants Localized Within 
B16 Melanoma Metastases 

Direct immunofluorescence utilizing FITC-conjugated anti- 

Met-72 MoAb binding on frozen sections of metastases have 

revealed Met-72 positive variants to be distributed as single 

cells and micrometastases within colonized organs. These 

poorly differentiated cells show a predominantly intense 

cytoplasmic pattern of staining and weaker cell membrane 

fluorescence (Figure 9 B, E) . Diffusely distributed 

micrometastatic foci of melanotic cells demonstrate a weaker 

cytoplasmic fluorescent signal (Figure 9 C) . Single cells and 

micrometastases have been observed in lungs colonized with 

B16-F1 or B16-F10 metastatic variants and in liver colonized 

with B16-F1 cells (Figure 9 E, F and G) . 

Specificity Characteristics of Syngeneic Antibodies 
Generated Against B16 Melanoma 

The kinetics of an IgG response against the poorly 

metastatic B16-F1 or JB/RH lines could be measured as early as 

8 days post immunization (Table 2) , with moderately higher 



21 
binding activity at 16 days (Table 3) . In most cases, 
increasing the immunizing dose above 10 5 cells had little 
effect on the levels of Ab produced. Antibody responses 
generated against either of the poorly metastatic lines (B16- 
Fl and JB/RH) cross-reacted with the other, while the highly 
metastatic clone was consistently negative in provoking an Ab 
response or in binding the Ab elicited by the other melanoma 
lines (Tables 2 and 3) . 

The ease and reproducibility of this anti-B16-Fl response 
prompted us to capture these reactivities by hybridoma 
technology, to further characterize the target cells and 
surface structures. Using established protocols (5,31), 
several MoAbs were obtained which demonstrated binding 
patterns in RIA very similar to anti-B16-Fl antisera. The 
population distribution of reactive cells was further analyzed 
by FACS. Specifically, anti-B16-Fl Abs were reacted against 
selected variants of C57BL/6 melanomas. The results of 6 
separate FACS experiments are typified by the example 
presented in Figure 10 A (antisera) and Figure 10 B (MoAb) , 
where Ab binding (green fluorescence) is plotted versus 
relative cell number. Antibody binding of anti-B16-Fl 
antisera or MoAb (stippled) was compared with eguivalent 
dilutions of NMS or control N-S.7 MoAb (solid line). The 
pattern of reactivity against the immunizing population (B16- 
Fl) is shown in Figure 10 A and 10 B, top left panels. A 
large proportion of the population shows greater fluorescence 



22 

than the NMS. In Figure 10 A, bottom left panel, the high 
lung colonizing line B16-F10 displayed a qualitatively similar 
binding pattern, the only difference being a much greater 
proportion of the cells were coincident with the negative 
fluorescent population than that seen with the poorly 
metastatic B16-F1. These differences were not apparent with 
anti-B16-Fl MoAb (Figure 10 B, bottom left) . When our most 
highly metastatic clone, BL6-10, was subjected to a similar 
analysis, only marginal reactivity with the anti-B16-Fl Ab was 
observed (Figure 10 A and 10 B, top right panels) . Finally, a 
recently derived C57BL/6 melanoma, JB/RH, which shows no 
experimental metastatic activity displayed the highest 
reactivity with the syngeneic anti-B16-Fl Ab (Figure 10 A and 
10 B, bottom right panels) . 

The second characteristic of Abs generated by these 
immunizations was the apparent melanoma specificity. As shown 
in Figure 11, anti-B16-Fl MoAbs reacted strongly with 
syngeneic, poorly metastatic melanomas (B16-F1 and JB/RH) , 
moderately with an allogeneic melanoma K1735, and were non- 
reactive with the other cells types in our tumor panel 
(fibrosarcoma, lymphomas, carcinoma, mastocytoma) or non- 
transformed murine fibroblasts or spleen cells. 

The third and most important characteristic of anti-B16- 
Fl Ab was the apparent inverse relationship between Ab binding 
and experimental metastatic potential of the cell lines and 
clones tested (Figure 11) . This was first suggested by the 



23 

reciprocal binding pattern of an anti-B16-Fl MoAb (2D8.3B1) 

versus our anti-metastatic, Met-72 MoAbs (5) (Figure 11) . 

Syngeneic Antibody Response Against a C3H Melanoma 

The binding of anti-B16-Fl MoAb against a moderately 

metastatic allogeneic melanoma line, K1735 (30,53), prompted 

us to examine the specificity of the Ab response to this C3H 

syngeneic melanoma. Experimental metastases of K1735 were 

generated and immune sera collected at necropsy. Reactivity 

against the immunizing line or allogeneic melanoma variants is 

shown in Figure 12 A. Similar to the Ab response against B16- 

Fl, this response syngeneic to a C3H, moderately metastatic 

melanoma line was seen to bind higher to the allogeneic, 

poorly metastatic variants. In addition, the reciprocal 

binding of anti-B16-Fl versus anti-Met-72 MoAb is apparent on 

the low versus high metastatic variants, respectively (Figure 

12 B) . 

Metastatic Potential of Melanoma Populations 
Negatively Selected By Syngeneic Anti-Melanoma Antibodies 

Strategies and methodology using immunomagnetic bead 

cellular affinity separation were developed to negatively 

select for the non-reactive cells in the heterogeneous 

immunizing B16-F1 population. Flow cytometric analyses of 

B16-F1 cells reacted against control (solid line) or immune Ab 

(stippled line) are shown in Figure 13. Anti-B16-Fl antisera 

(Figure 13 A) or MoAb (Figure 13 B) binding is compared to 

non-reactive populations, obtained after two cycles of 

immunomagnetic bead separation, restained with anti-B16-Fl 



24 
antisera (Figure 13 C, F) or anti-B16-Fl MoAb (Figure 13 D, 
E) . The efficiency of the immunomagnetic bead separation 
protocol is apparent in panels C, D, and E. 

The FACS profiles of B16-F1 and B16-F10 cells before and 
after application of the immunomagnetic bead separation 
procedure are shown in Figure 14. Negatively selected cells 
obtained by anti-B16-Fl depletion were compared with cells 
similarly fractionated with NMS or control N-S.7 MoAb. A 
portion of cells from each of these separations was 
immediately tested for experimental metastatic activity. As 
shown in Figure 15, the experimental metastatic potential of 
control Ab and syngeneic anti-B16-Fl Ab fractionated cells was 
distinctly different. These results are in agreement with the 
anticipated doubling in metastatic activity since 
approximately 60% of the melanoma population was removed by 
specific Ab + immunomagnetic bead separation. Negligible and 
significant differences in pulmonary colonization were 
observed between control and anti-B16-Fl Ab treated B16-F1 and 
B16-F10 populations, respectively (Table 4) . In addition, the 
fractionation procedure was seen to impact significantly on 
the extent of extrapulmonary metastases, morbidity, and 
mortality when compared to controls. As seen in Figure 15, 
there is a clear trend toward more aggressive extrapulmonary 
colonization with both the B16-F1 and B16-F10 negatively 
selected populations. In particular, the involvement of lymph 
nodes was statistically significant, by analysis of variance, 



25 

in the cases of B16-F10 cells negatively selected with 

antisera (p=0.0196) and MoAb (p=0.0103) (Table 4). 

Biochemical Characterization of Anti-B16-Fl Reactive Species 

Western blot analysis comparing antisera and MoAb 

reactivity against detergent lysates of the poorly metastatic 

lines B16-F1 and JB/RH, versus the highly metastatic B16 

clone, BL6-10, is shown in Figure 16. The anti-B16-Fl 

antisera immuno-stains two distinct bands of Mr 26,000 and 

45,000 and a diffuse region between Mr 69,000 to 200,000 with 

both of the poorly metastatic melanoma lines, B16-F1 and JB/RH 

(Figure 16 lane b) . Two of the anti-B16-Fl MoAb, 2D8.3B1 and 

1F11.1E12, identify a corresponding Mr 45,000 band in lysates 

from the poorly metastatic melanoma lines (Figure 16 lane c 

and d) . A less intensely stained band of Mr 45,000 has been 

identified using the same anti-B16-Fl MoAb by Western blot 

analysis of B16-F10 and K1735 detergent lysates (data not 

shown). In addition, a Mr 50,000 band was stained with the 

same anti-B16-Fl MoAb in the JB/RH lysate. As anticipated 

from cell binding assays of the syngeneic antisera, the highly 

metastatic clone BL6-10 did not reveal any specific bands 

(Figure 16, lanes b, c, d) . 

In Situ Characterization of Metastatic Variants Within 
Experimental Metastases and Subcutaneous Masses 

of B16 Melanoma 

The cellular morphology of metastatic variants expressing 

Met-72 or anti-B16-Fl antigen was evaluated by 

immunocytochemical analysis of touch preparations of freshly 



26 
excised metastases. Impression smears of liver, lymph node 
and lung metastases derived experimentally from B16-F1 and 
BL6-10 cells were examined. A lymph node metastasis derived 
experimentally by injection of BL6-10 is shown in Figure 17. 
Variably melanotic cells stained most positively with anti- 
Met-72 MoAb (Figure 17 B) with individual cellular 
heterogeneity evident for anti-Met-72 MoAb and anti-B16-Fl 
MoAb (Figure 17 C) binding. Most cells exfoliated from BL6-10 
derived metastases demonstrated background levels of anti-B16- 
Fl MoAb binding with an occasional (less than 1%) strongly 
positive cell. 

Previously, analyses of cytospin preparations of B16 
melanoma ovarian metastases had revealed cellular 
heterogeneity in Met-72 expression (54) . Analysis of frozen 
sections of experimental metastases revealed similar levels of 
cytoplasmic and membrane heterogeneity within metastases to 
the ovary (Figure 18 B) and greater omentum (not shown) when 
stained using anti-Met-72 MoAb. Anti-B6-Fl MoAb generally 
showed less intense staining, with an occasional brightly 
positive cell (less than 1%) . Frozen sections of lungs were 
analyzed using the immunoperoxidase technique for comparison 
with the earlier direct immunofluorescence studies. Again, 
Met-72 positive cells were variably melanotic and stained 
brightly as individual cells or microscopic foci within the 
parenchyma. The majority of melanotic cells within larger 
metastatic foci showed weak cytoplasmic staining (Figure 19 C, 



27 
D) . Interestingly, anti-B16-Fl MoAb revealed strong 
cytoplasmic and membrane staining within the larger pulmonary 
metastatic foci (Figure 19 E, F) . 

To address the effect of the host immune response on the 
emergence of metastatic variants, B16 melanoma variants were 
localized within developing s.c. tumors maintained in normal 
or sublethally irradiated hosts. B16-F1 or BL6-10 cells were 
injected s.c. into normal or 500 Gy irradiated syngeneic mice 
and into Balb/c nu/nu mice (2 mice per group) . Resulting 
masses were removed after 12 days, measured in three 
dimensions to calculate a mean tumor volume (Table 5) and snap 
frozen. Serial cryostat sections (6 to 12 jim) of each mass 
from each animal were examined by ABC immunoperoxidase 
analysis as described. Monoclonal antibody binding was 
assessed on a scale of negative (lowest) to 5+ (highest) and 
is summarized in Table 5. Anti-Met-72 MoAb binding was 
clearly evident within regions of tumors and on variably 
melanotic cells within normal syngeneic hosts (Figure 20 C and 
D) as had been noted previously (54) . Anti-B16-Fl MoAb 
binding to serial sections of the same masses clearly showed 
no reaction within the regions which were most brightly Met-72 
positive (Figure 20 E and F) . The most striking observation 
was the increase in the number of Met-72 positive cells and 
the increase in the level of anti-Met-72 MoAb binding per cell 
throughout the masses in 500 Gy irradiated syngeneic hosts 
(Figure 21 C, D and G) . This increase was much more apparent 



28 
in tumors induced by BL6-10 cells than in those induced by 
B16-F1 cells. This difference was also more apparent in 
sublethally irradiated, syngeneic hosts than in allogeneic 
athymic hosts (Figure 22 C and D) . There was a slight 
increase in the level of anti-B16-Fl MoAb binding per cell 
within tumors induced by BL6-10 cells maintained in irradiated 
syngeneic or allogeneic athymic hosts (Table 5, Figures 21 and 
22, E and F) . 
Immunohistology of Syngeneic Healing Skin Wounds and Embryos 

Surgically created skin wounds of C57BL/6 mice were 
examined by ABC immunoperoxidase analysis of serial cryostat 
sections (6 /xm) during early, mid, and late phases of healing. 
Anti-Met-72 MoAb reactivity was not detected in 24, 48, 72, or 
9 6 hour healing wounds (data not shown) . 

Embryos of C57BL/6 mice were removed at day 11 and 15 of 
gestation and snap frozen. Immunohistology of serial cryostat 
sections (12 /xm) were examined from 2 embryos at each time 
point. Neither anti-Met-72 MoAb nor anti-B16-Fl MoAb 
(2D8.3B1) binding was detected at either time point during 
gestation (data not shown) . 



29 



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monoclonal antibody. 

Affinity purified anti-Met-72 MoAb binding to BL6- 
10 cells analyzed by FACS indirectly using FITC-SAM 
(panel A) . Biotin was reacted with affinity 
purified anti-Met-72 MoAb at three biotin ester to 
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33 



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Figure 3. Retention of binding specificity of anti-Met- 
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Various amounts of biotin conjugated anti-Met-72 
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melanoma clone, BL6/10, the poorly metastatic B16 
melanoma parent, B16-F1, and a recently derived, 
poorly metastatic C57BL/6 melanoma, JB/RH. The 
extent of specific binding was measured after the 
addition of 125 i labeled streptavidin. Values are 
expressed as the mean of triplicate determinations 
of cpm bound ± S.D. 



35 



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(simn eA|;B|9J) 13NNVH0 d3d INflOO 



Figure 5. Clones derived from experimental metastases to 
ovaries retain a high expression of Met-72 
upon repeated cycling in vivo . 

a. in vivo cycle number for the selection of 
metastatic variants with ovary specificity. 

b. number of mice per group. 

c. number of mice with metastases to organ site. 

d. Binding of anti-Met-72 MoAb to ovarian 
metastatic variants as detected by RIA. Results 
are expressed as a binding index, which is 
calculated by dividing the mean cpm 125 i pA 
bound with anti-Met-72 MoAb divided by the mean 
cpm 125 i pA bound with N-S.7 MoAb (background 
binding) . 

e. Other = kidney, mesentery. 



39 



Selection Metastases 

Group cycle „b to 



Primary 
Ovarian 

Metastasis 
0-1 



Clone 0-1.1 
of Ovarian 
Metastasis 



0-1.2 



0-1.3 



8 



19 



23 



8 



Lungs 



Ovary 

Bone 
Marrow 

Lymph 
Node 

Other e 



V/////////////777A 



% of Mice with Metastases 
20 40 60 80 



Met-72 
Expression 



i 



Y/////////////////////////77. 



A 



7m 



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'/////////A 



77777* 



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Bone 
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Lymph 
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////////////////////A 



V////////////ZA 



- 40 



20 



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Figure 7. Met-72 positive variants of B16 melanoma 
detected in cytospin and impression smear 
preparations of metastases. 

Cell suspensions from a fresh ovarian metastasis 
were stained using biotinylated control N-S.7 MoAb 
(A) or anti-Met-72 MoAb (B) . Impression smears of 
experimental lung metastases (C, D) or spontaneous 
lung metastases (E, F) were stained using biotin 
conjugated N-S.7 MoAb (C, E) or anti-Met-72 MoAb 
(D, F) . (A - F X 270) . 



43 







J 





I 




• • 








Figure 8. Localization of Met-72 positive variants 

within developing B16 subcutaneous melanoma. 

Biotin conjugated N-S.7 MoAb bound to a cryostat 
section of a B16-F1 subcutaneous melanoma (A) x 55 
and (B) x 135. Biotin conjugated anti-Met-72 MoAb 
bound to a serial section of the same B16-F1 s.c. 
melanoma x 55 (C) , x 135 (D) , x 1350 (E) , x 550 
(F). 



45 




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1 





Figure 10. Flow cytometric analysis of anti-B16-Fl 
immune sera (A) and anti-B16-Fl MoAb 
(2D8.3B1) (B) binding to C57BL/6 melanomas. 

The solid line indicates control Ab binding; the 
stippled line indicates anti-B16-Fl Ab binding. 
The antisera is from the same individual mouse 
(challenged i.v. with 3 X 10 5 B16-F1 cells) which 
was used to generate the anti-B16-Fl hybridoma, 
2D8. Log green fluorescence is on the x axis; 
relative cell number per channel on the y axis. 



53 



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59 






Figure 14. Flow cytometric analysis of B16 melanoma 
before and after immunomagnetic bead 
selection and removal of reactive 
subpopulations . 

B16-F1 and B16-F10 cells reactive with NMS (solid 
line) or anti-B16-Fl antisera (stippled line) 
before and after immunomagnetic bead depletion were 
analyzed by FACS after FITC-SAM binding. Log green 
fluorescence is on the x axis; relative cell 
number per channel (maximum =350) on the y axis. 



61 



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Figure 16. Western blot analysis of anti-B16-Fl immune sera 
and MoAb binding to lysates of syngeneic melanoma 
metastatic variant cell lines. 

A 10% polyacrylamide gel was western blotted as described. 
Apparent molecular weights of marker proteins (Mr X 10" 3 ) 
are indicated on the abcissa. 

a. Normal mouse serum. 

b. Anti-B16-Fl antisera. 

c. Anti-B16-Fl MoAb, 1F11.1E12. 

d. Anti-B16-Fl MoAb, 2D8.3B1. 

e. Anti-B16-Fl MoAb, 2H5.1B1. 

f. Anti-sheep RBC MoAb, N-S.7. 



Figure 17. Immunocytology of metastatic variants 

exfoliated from fresh experimental lymph node 
metastases of BL6-10. 

Impression smears of expermental lymph node 
metastases of BL6-10 were processed by the ABC 
immunoperoxidase method as described. The level of 
staining generated by N-S.7 MoAB (A) is compared 
with anti-Met-72 MoAb (B) and anti-B16-Fl MoAb, 
2D8.3B1 (C) (A, B and C X 1350). 



67 




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69 




w 




Figure 19. Localization of B16 melanoma variants within 
experimentally colonized lungs. 

Mice were injected i.v. with 3 X10 5 B16-F1 cells 
per 0.2 ml PBS; colonized lungs were removed and 
snap frozen at necropsy 21 days later. Serial 
cryostat sections (12 fim) were analyzed by the ABC 
immunoperoxidase technigue as described. Control 
(N-S.7) binding is shown in A and B; anti-Met-72 
MoAb binding in C, D and G; and anti-B16-Fl MoAb 
(2D8.3B1) binding in E and F. (A, C and E x 135; B, 
D, F and G x 1350) . 



71 




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Figure 20. Localization of B16 melanoma variants within 
subcutaneous tumors maintained in normal 
syngeneic hosts. 

A s.c. mass induced by injection of 2 X 10 6 BL6-10 
cells per 0.1 ml PBS into a normal C57BL/6 mouse 
was excised after 12 days and snap frozen. Serial 
cryostat sections (12 jim) were examined by the ABC 
immunoperoxidase technique as described. Control 
N-S.7 MoAb binding is shown in A and B; anti-Met-72 
MoAb in C and D; and anti-B16-Fl MoAb (2D8.3B1) in 
E and F. (A, C and E x 135; B, D and F x 1350) . 



74 




Figure 21. Localization of B16 melanoma variants within 
subcutaneous tumors maintained in 500 Gy 
irradiated syngeneic hosts. 

A C57BL/6 mouse was exposed to 500 Gy irradiation 
and injected with 2 X 10 6 BL6-10 cells per 0.1 ml 
PBS on the same day. A s.c. mass was excised after 
12 days, snap frozen and serial cryostat sections 
(12 Mm) were examined by ABC immunoperoxidase 
analysis as described. Control N-S.7 MoAb binding 
is shown in A and B; anti-Met-72 MoAb in C, D and 
G; and anti-B16-Fl MoAb (2D8.3B1) in E and F. (A, 
C and E x 135; B, D, F and G x 1350). 






76 







\?Awxmim£ '^ 







Figure 22. Localization of B16 melanoma variants within 
subcutaneous tumors maintained in allogeneic 
athymic hosts. 

A s.c. mass induced by injection of 2 X10 6 BL6-10 
cells per 0.1 ml PBS into a Balb/c nu/nu mouse was 
excised after 12 days and snap frozen. Serial 
cryostat sections (12 urn) were examined by the ABC 
immunoperoxidase technique as described. Control 
N-S.7 MoAb binding is shown in A and B; anti-Met-72 
MoAb in C and D; and anti-B16-Fl MoAb (2D8.3B1) in 
E and F. (A, C and E x 135; B, D and F x 1350). 



78 




DISCUSSION 

These studies have focused on 1) the localization of B16 
melanoma metastatic variants within developing s.c. tumors and 
metastases and 2) the effect of the tumor bearer's humoral 
immune response on the regulation of the levels of metastatic 
variants within developing B16 melanomas. 

The significant observations were first, that Met-72 
expression is discretely localized in situ in the primary B16 
melanoma mass. Met-72 positive variants are found to be 
variably melanotic cells regionally located within developing 
s.c. tumors grown in normal syngeneic hosts. Secondly, Met-72 
expression is enriched on experimentally derived metastases. 
As increased ovarian colonizing potential is generated, Met-72 
expression of melanoma cells derived from metastases to 
ovaries is enriched. There is individual cellular 
heterogeneity in the level of expression of Met-72, as 
visualized by immunocytology of metastases, with small foci 
of cells being the most positive within colonized organs. 
Thirdly, melanoma lines with poor experimental metastatic 
activity consistently elicit strong syngeneic Ab responses, 
while highly metastatic clonal variants do not. This Ab 
response can be used to subdivide metastatic from 
nonmetastatic cells present within the immunizing population. 

79 



80 
Finally, the Ab response to poorly metastatic variants 
detects uniformly distributed variants within larger 
metastatic foci in experimentally colonized lungs. 

The existence of subpopulations of cells exhibiting a 
range of metastatic potential within heterogeneous tumors has 
been substantiated in a number of systems (3,55). In the 
original studies leading to the present work, a strong 
correlation between the quantitative expression of a Mr 72,000 
glycoprotein (Met-72) and experimental metastatic activity of 
over 30 in vitro grown B16 melanoma clones was demonstrated 
(5,11). Flow cytometric analysis and cell sorting procedures 
using anti-Met-72 MoAb have directly shown that high levels of 
Met-72 expression is characteristic of cells with a high 
experimental metastatic potential (12) . Our current studies 
were designed to determine the potential utility of anti-Met- 
72 MoAb to visualize and localize Met-72 positive metastatic 
variants within progressing and metastatic B16 melanoma 
masses. 

Results of the experiments reported here greatly expand 
our previous knowledge of Met-72 antigen expression and its 
correlation with metastatic potential in vivo. Localization 
of its expression in primary s.c. tumors is notably discrete, 
and not randomly distributed throughout the developing tumor 
mass (Figures 8 and 20) in immunologically normal syngeneic 
hosts. Immunohistologic examination of progressing tumors, 
excised at sequential times during s.c. growth, shows a 



81 
recurrent pattern of localization of Met-72 positive variants. 
These variably melanotic cells are seen within regions of the 
masses, the most brightly positive cells within smaller 
satellites. The implications of this selective localization 
are twofold. First, induction of Met-72 positive variants may 
be controlled by microenvironmental factors. Secondly, even 
if random somatic mutational events yield single metastatic 
variant cells within a solid tumor mass, their outgrowth into 
colonies may be directed by chemotactic factors which are 
microenvironmentally determined. These observations suggest 
that microenvironmental influences may function regionally to 
influence metastatic potential. Our observations in the B16 
melanoma model of metastasis are consistent with those of 
Gabbert, et al. (56) . They suggest that morphologic 
transitions at the invading front of rat malignant carcinoma 
may signify a localized process of tumor de-differentiation. 
Tumor cell locomotion may be specifically enhanced in regions 
observed to have a loss of basement membrane and decreased 
numbers of desmosomes between tumor cells. Clearly, the 
interactions in tumor microregions greatly influence tumor 
heterogeneity, as recently reviewed by Sutherland (57) . 

An important aspect of these studies focused on isolation 
and visualization of Met-72 positive variants within 
metastatic foci of B16 melanoma. The sophisticated 
capabilities of the fluorescence activated cell sorter have 
provided evidence that Met-72 expression may be a common 



82 
surface phenotype of B16 melanoma metastatic variants, 
irrespective of their organ colonization after i.v. 
inoculation. Experimentally induced ovarian metastases were 
directly shown to express Met-72 (54) , as has been reported 
for experimental lung metastases of the B16 melanoma (12) . As 
previously demonstrated with lung colonizing melanoma cells 
(58,59), ovarian colonizing variants were selected by repeated 
in vivo cycling. These variants showed increased levels of 
Met-72 positivity (Figure 5) . Clones of bone marrow derived 
metastases also demonstrated increased levels of Met-72 
expression (Figure 6) . Clones derived from B16-F1 metastases 
to the liver, heart, lymph node and stomach wall have all 
demonstrated increased Met-72 antigen expression which is 
maintained after repeated in vitro passage. 

Rapid visualization of individual cellular expression of 
Met-72 was achieved by two immunocytologic methodologies: 1) 
cytospin preparations of experimental ovarian metastases and 
2) impression smears of experimental and spontaneous lung 
metastases. The ease of impression smear immunocytochemistry, 
especially, has permitted rapid characterization of the 
surface phenotype of cells dislodged from colonized lungs. 
Primary melanomas and other well encapsulated masses do not 
present suitable specimens for impression smears. 

The significant findings of these studies are that (1) 
Met-72 expression is discretely localized in situ in the 
primary B16 melanoma mass, and (2) cell surface expression of 



83 
Met-72 may prove to be a generalized phenomenon of melanoma 
metastatic variant populations, regardless of organ 
selectivity. The study of metastatic tumor cell evolution 
influenced by 1) interactions with tumor derived elements and 
2) host selection pressures during tumor progression has 
eluded tumor biologists. The ability to isolate metastatic 
variant cells from primary tumor tissue may enable us to 
guantitate their presence and evaluate their differences 
during tumor progression and metastatic outgrowth. 

With few exceptions, humoral immunity towards syngeneic 
tumors has been generally regarded as beneficial for host 
survival (4,6,55,57,60). This belief, and the availability of 
therapeutic quantities of "tumor reactive" MoAb has resulted 
in a number of clinical trials for the treatment of human 
cancer. 

The notion that poorly immunogenic variants exist within 
tumors has been supported primarily by the isolation of 
subpopulations of tumor cells which are resistant to cellular 
effector systems in vitro (61-64) . A major question left 
unanswered by these studies is the extent to which humoral 
immunity may contribute to the changing proportion of 
metastatic variants within tumor populations. Previous 
studies have not attempted to correlate syngeneic Ab responses 
against B16 melanoma-associated antigens with spontaneous or 
experimental metastatic activity of melanoma subpopulations 
(14-16,20,65,66). Some studies have indirectly correlated 



84 
syngeneic anti-B16 melanoma responses with metastatic activity 
(13,17,18) or have found no correlation between metastatic 
potential of clones derived from B16 melanoma and syngeneic Ab 
responses induced by s.c. immunization with those clones (19) . 
McDonald, et al. (67) reported syngeneic Ab detected in sera 
of mice challenged with the highly invasive variant of B16 
melanoma, B16-BL6. However the subpopulation and functional 
specificity of these Ab was not determined. 

The ease and frequency with which host anti-melanoma 
humoral immunity can be demonstrated in both experimental and 
clinical situations has prompted us to examine whether Ab 
responses in these situations may in fact be targeted against 
functionally distinct subsets of melanoma. While studies 
directly correlating metastatic activity and immunogenicity of 
melanoma subpopulations have not been described previously, 
several lines of investigation have suggested the possibility 
that poorly metastatic subpopulations are the primary Ab 
targets. First, syngeneic and autochthonous Ab responses 
against melanoma are frequently observed (13-20,65-67). 
However, detection of syngeneic Ab reagents defining highly 
metastatic melanoma variants often require manipulation of 
host immunity to override these primary responses (5) . 
Second, Ab against human melanoma, obtained by xenogeneic 
immunization, are most reactive with the more differentiated, 
benign melanoma forms (68-71) . Recently, Herd reported the 
production and characteristics of MoAb from syngeneic mice 



85 
bearing B16 melanoma masses (72) . Some of the MoAb were 
reported to enhance or inhibit B16-F1 lung colonization and at 
the same time to decrease lung colonization of the high lung 
colonizing variant, B16-F10. Recent studies by others within 
our laboratory now suggest that in addition to the primary 
host responses against subpopulations of poorly metastatic B16 
melanoma, the isotype of these antibodies has a dramatic 
biologic consequence (Kimura et al. , unpublished results) . 
Mice challenged i.v. with syngeneic B16-F1 or BL6-10 cells 
after i.p. treatment with the IgG 3 isotype switch variant of 
anti-Met-72 MoAb were observed to have increased numbers of 
lung nodules. 

While it is true that syngeneic anti-melanoma MoAbs which 
interfere with experimental metastasis can be isolated 
(5,65,72), these Ab responses are rare. In these cases 
multiple immunizations, with or without adjuvant, coupled to 
highly specific screening procedures are needed to isolate 
these reactivities. The major finding to emerge from our 
study is that melanoma lines with poor metastatic activity 
consistently elicit strong syngeneic Ab responses, while 
highly metastatic clonal variants do not. These anti- 
nonmetastatic Ab activities are readily demonstrated by 
analyzing the serum from melanoma immunized mice. Poorly 
metastatic melanoma lines such as B16-F1 and JB/RH elicit Ab 
responses which can be used to further subdivide metastatic 
from non-metastatic cells present within the immunizing 



86 
population. This conclusion is based on 1) Ab binding to well 
characterized melanoma lines and clones shown by RIA, FACS, 
and western blot analysis, and 2) experimental metastatic 
capabilities of negatively selected cells. 

The results of our immunomagnetic bead depletion assays 
show a total increase in the extent of experimental metastases 
generated after the removal of anti-B16-Fl reactive 
subpopulations. This increase is subtle with the 
heterogeneous parental B16-F1 line, but significant with the 
B16-F10 line. It is not unexpected that a subpopulation of 
anti-B16-Fl antisera reactive cells remains after removal of 
the anti-B16-Fl MoAb reactive cells (Figure 13 F) . Our 
results support the suggestion that B16-F10 was selected for 
increased lung colonization potential, but that organ 
selectivity is not absolutely eguivalent to increased 
metastatic potential. B16-F1 and B16-F10 are heterogeneous 
lines with respect to many phenotypes (6) . Metastases 
generated from B16-F1 and B16-F10 may maintain heterogeneity 
with regard to organ colonization potential. Our findings are 
consistent with the interpretation that non-metastatic cells 
present within the parental B16-F1 line (and at lower levels 
in the selected B16-F10 line) elicit a strong and selective Ab 
response; the negative binding cells representing the more 
highly metastatic variants. Removal of the poorly metastatic 
variants from the total population allows the extrapulmonary 
colonization potential to become more evident. This is more 



87 
so with B16-F10, because on a cell to cell basis this 
heterogeneous population contains more cells with a more 
efficient metastatic potential. 

The mechanism of this extrapulmonary colonization may be 
suggested by the results of experiments by Nakajima, et al. 
(73). Metastatic variants of B16 melanoma, including B16-F1 
and B16-F10, were shown to differ from each other in their 
ability to degrade heparan sulfate which may be related to 
their ability to adhere and grow in specific organs (73) . 
Specificity of adhesion between murine tumor cells and 
microvascular endothelium from different organs has been 
demonstrated by Auerbach et al. (74) . Thus, variants 
remaining after depletion of anti-B16-Fl reactive 
subpopulations may mainfest their adhesive phenotypes more 
efficiently. 

The influence of host immunity on the induction or 
dissemination of metastatic variants appears variable 
depending upon which of the heterogeneous phenotypes reguired 
for tumor progression one is attempting to study (1-4) . The 
studies reported here strongly suggest the relevance of 
comparing metastatic variant clonal lines with heterogeneous 
lines. Caution must be exercised when interpreting results 
obtained using heterogeneous lines which appear homogeneous 
with regard to one phenotype, such as B16-F10 cells. 

Anti-B16-Fl antisera and MoAb have recently been used to 
immunoprecipitate from detergent lysates of melanoma cells 



88 
biosynthetically labeled with [ 3 H] -leucine, bands which 
correspond to those identified by western blot analyses (Mr 
32,000, 45,000 and 55,000 on 5 to 15% or 10% acrylamide slab 
gels under reducing conditions, Parratto & Kimura, unpublished 
results) . Molecular weight characteristics of anti-B16-Fl 
reactive species bear some resemblance to those reported by 
others (14-16,20,75-79). However, surface expression of those 
markers versus metastatic behavior of the cells was not 
determined in those studies. The Mr 45,000 band identified by 
the anti-B16-Fl antisera or corresponding MoAb cannot be 
against normal Class I antigens of the H-2 complex, as there 
is no measurable binding to C57BL/6 spleen cells or other 
syngeneic tumor cell lines. 

Additional immunohistologic analyses of cells isolated 
from and localized within developing B16 melanoma metastases 
revealed several interesting binding profiles. First, 
impression smears revealed an increased level of anti-Met-72 
MoAb binding to individual cells exfoliated from the parietal 
lung surface compared to cells exfoliated from cut surfaces of 
well encapsulated extrapulmonary metastases. On the other 
hand, anti-B16-Fl MoAb binding was weak to cells exfoliated 
from these same metastases. In addition, cryostat sections of 
colonized organs showed intensely Met-72 positive variants to 
be variably melanotic individual cells or cells within small 
metastatic foci. However, anti-B16-Fl MoAb showed a diffuse 
pattern of reactivity with melanotic cells within larger B16- 



89 
Fl pulmonary metastases and moderate binding to 
extrapulmonary metastases. A morphologic assessment of B16 
metastases within the syngeneic host has been reported 
recently by Dingemans (80) . Patterns of metastatic growth 
within experimentally colonized liver and lung were compared 
revealing small, dense tumor cell nodules within encapsulated 
spheres (liver) or flat, ill defined foci (lung) . This was 
interpreted as apparent regions of focal proliferation 
alternating with active movement and was explained on the 
basis of interactions with extracellular matrix. Our studies 
show that the Met-72 positive variants are observed within 
microscopi foci of colonized organs. These observations 
suggest that Met-72 antigen expression may be correlated to 
cells within regions of focal proliferation. 

Our results localizing B16 melanoma variants within 
developing s.c. masses and metastases are in accordance with 
the observations of Yamasaki, et al. (81) . Distinctly 
different regional distributions of radiolabeled syngeneic 
MoAb directed against B16 melanoma was observed in vivo in B16 
melanoma masses. These patterns of distribution were 
attributed to differentiation stages of melanoma cells, 
however, MoAb binding was not correlated with metastatic 
potential in their study. 

Analysis of metastatic variant localization within s.c. 
tumors maintained in syngeneic, sublethally irradiated or 
allogeneic, athymic hosts revealed a striking observation. 



90 
The number of Met-72 positive variants and their individual 
level of Met-72 expression was increased in s.c. masses 
maintained in irradiated or immunocompromised hosts. This 
observation may contradict the contention that the host Ab 
response is directed against poorly metastatic variants. 
Clearly these preliminary experiments warrant further 
investigation . 

The basic biologic and practical implications of these 
findings suggest that host humoral immunity to melanoma may 
represent a major driving force favoring tumor progression and 
metastasis. Non-immunogenic subpopulations of melanoma of 
higher metastatic potential could thus continue to emerge and 
become the dominant cell type in the absence of other 
selective forces. Thus, anti-tumor MoAb raised by similar 
immunization protocols for therapeutic studies should first be 
used to examine the metastatic competence of the target 
population. Additional criteria besides "melanoma-specific" 
may be reguired and warranted, before passive immunotherapy 
involving MoAb is applied to clinical situations. Additional 
studies involving host humoral immunity and tumor progression 
are clearly warranted. 



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BIOGRAPHICAL SKETCH 
I was born on April 24, 1956, in Philadelphia, 
Pennsylvania. My elementary education was in the public 
schools of Philadelphia; secondary education in Upper Dublin 
Township, Pennsylvania. I received a B.S. in biology from 
Villanova University, Villanova, Pennsylvania, May, 1978, and 
my D.V.M. from the University of Florida, Gainesville, 
Florida, May, 1985. My professional work experience has been 
in the area of veterinary medicine as an assistant from May, 
1975, to December, 1978 with private practitioners in 
Pennsylvania. I hold a current license to practice veterinary 
medicine in Florida and have been a part time associate or 
relief veterinarian from August, 1985, to the present with 
private practitioners in Florida. My graduate research 
experience has been focused in the area of tumor immunology 
and cancer biology. As a graduate research assistant in the 
Department of Pathology, College of Medicine, University of 
Florida, I have trained with Dr. Richard T. Smith and Dr. 
Byron Croker. My dissertation mentor has been Dr. Arthur K. 
Kimura. I was awarded an NIH Traineeship in the Immunobiology 
of Cancer from the Fall of 1987 through the Fall of 1988; am 
named in Who's Who in Veterinary Science and Medicine, First 
Edition, 1987-88; and have received awards for service from 

100 



101 
the President of the University of Florida, 1985, the Student 
Chapter of the American Veterinary Medical Association, 1985, 
and the American Veterinary Medical Association Auxiliary, 
1984. I am a current member of the American Veterinary 
Medical Association and the American Association for the 
Advancement of Science. The research embodied in my 
dissertation has been presented as abstracts to three national 
scientific meetings and has been published and submitted for 
publication in refereed journals. I was offered post-doctoral 
fellowships for further training in cancer biology at Mount 
Sinai Research Institute, Toronto, Ontario, Canada; Scripps 
Clinic and Research Institute, La Jolla, California; the 
Medical College of Virginia, Massey Cancer Center, Richmond, 
Virginia; and the Department of Dermatology, New York 
University Medical School. I have accepted a position as 
staff scientist with a biotechnology corporation in 
Charleston, South Carolina, and as Adjunct Assistant 
Professor, Department of Laboratory Medicine and Immunology, 
Medical University of South Carolina. 



I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for 
the deqree of Doctor of Philosophy. 

Arthur K. Kimura, Chair 
Associate Professor, Pathology 
and Laboratory Medicine 

I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for 
the degree of Doctor of Philosophy. 




Richard T. Smith 
Professor, Pathology 
and Laboratory Medicine 



I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for 
the degree of Doctor of Philosophy. 




K. Kendall - Pier son 
Professor, Pathology 
and Laboratory Medicine 

I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for 
the degree of Doctor of Philosophy. 

Sigurd Normann 
Professor, Pathology 
and Laboratory Medicine 

I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for 
the degree of Doctor of Philosophy. 




'<&<? 



Maron B. Calderwood-Mays 
Associate Professor, Pathology 
and Veterinary Medicine 



I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for 
the degree of Doctor of Philosophy. 

lU^~ (a)aJt 

Chris West 

Associate Professor, Anatomy 

and Cell Biology 

This dissertation was submitted to the Graduate Faculty of 
the College of Medicine and the Graduate School and was accepted 
as partial fulfillment of the requirements for the degree of 
Doctor of Philosophy. 

December 1988 &*£?!»* ***^ 

Dean, College of Medicine 

Dean, Graduate School